The objective of the proposed research is to gain increased understanding of the range and mechanism of responses elicited in target cells by 17 beta-estradiol. The human breast cancer cell line MCF-7, which contains receptors for estrogen, androgen, progestins and glucocorticoids, will be used as a model system. We will study the early response of these cells to estrogen by measuring changes in the rate and type of RNA and protein synthesis, changes in the levels of cAMP and cGMP and alterations in phospholipid metabolism. We will then enucleate cells using cytochalasin B and determine whether the enucleated cells are capable of any of these responses to estrogen. Mutants of MCF-7 with altered sensitivity to estrogen will be obtained by selecting clones which are resistant to the antiestrogen Tamoxifen. Special emphasis will be placed on trying to select temperature sensitive mutants. We will also determine, in cultures of synchronized MCF-7 cells, whether the nature of the response to estrogen varies with the phase of the cell cycle and will determine when in the cell cycle the estrogen receptor is synthesized and when, following DNA synthesis, the new chromatin develops acceptor activity for the receptor.